Project Summary A typical virus capsid consists of hundreds of copies of capsid protein that act as the protective package of the viral genome. Therefore, the typical capsid assembly reaction will have hundreds of steps, each one of which can go wrong. Yet, even in vitro, self-assembly of virus capsids can occur spontaneously and with high fidelity. This reaction is of fundamental interest to virologists, a focus of antiviral development, and a general model of self-assembly with implications for harnessing viruses for nano- and biotechnology. Understanding the mechanism of virus assembly requires not only knowledge of precursors and final products, but also access to intermediates. Where many rare intermediates are involved, ensemble methods obscure them so that virus assembly resembles a two-state reaction, i.e., only subunits and capsids are observed. Computational models of assembly suggest that the observed kinetics reflect establishment of early intermediates needed to support capsid formation. The nucleation step and these early intermediates are believed to play a role in recruiting viral components in vivo. In our previous work, we established hepatitis B virus (HBV) assembly as a well-defined and robust experimental system for interrogating assembly reactions. HBV capsids, composed of 120 homodimers, self-assemble in response to buffer conditions. Surprisingly, we found metastable intermediates in assembly and disassembly. These results are timely as HBV capsid assembly has become an important target for development of antiviral assembly effectors which over- stimulate nucleation, distorting the distribution of intermediates and often their structure. Resistive-pulse sensing on in-plane nanofluidic devices is a unique platform and permits a label-free, single-particle approach to monitor assembly in real time at biologically relevant concentrations (nM to M). Our resistive-pulse measurements have provided highly complementary data to other state-of-the-art techniques, e.g., time-resolved small angle x-ray scattering, light scattering, charge detection mass spectrometry, and transmission electron microscopy. All of these approaches require much higher protein concentrations than resistive-pulse sensing and, thus, obscure many features of these complex reactions. We have developed needed fabrication methods, characterized individual HBV capsids, and monitored their assembly below, near, and above the pseudo-critical dimer concentration. Because of our ability to probe single particles in real time and over a range of assembly conditions, we are now poised to address a number of questions, previously thought unanswerable. The specific aims for this application are to: (1) integrate on- device mixing and multiplexed detection to probe early time points of assembly; (2) compare assembly of virus capsids with and without assembly effectors; (3) evaluate capsid assembly and disassembly in the presence of chaotropes; (4) monitor the evolution of incomplete particles; and (5) fabricate nanoimprinted in- plane nanofluidic devices for assembly and disassembly experiments.